Method of analysing a blood sample of a subject for the presence of a foetal disease or condition marker

Abstract

The present invention relates to a method of analysing a blood sample of a subject carrying a foetus for the presence of a foetal disease or condition marker, said method comprising the steps of a) extracting nucleic acid from anucleated blood cells, preferably thrombocytes, in said blood sample to provide an anucleated blood cell-extracted nucleic acid fraction, and b) analysing said anucleated blood cell-extracted nucleic acid fraction for the presence of a foetal disease or condition marker, wherein said foetal disease or condition marker is a nucleic acid of a foetus, or wherein said foetal disease or condition marker is a foetal disease or condition-specific expression profile of genes of a cell of said foetus.

1 . A method of analysing a blood sample of a subject carrying a foetus for the presence of a foetal disease or condition marker, said method comprising the steps of
 a) extracting nucleic acid from anucleated blood cells, preferably thrombocytes, in said blood sample to provide an anucleated blood cell-extracted nucleic acid fraction, and   b) analysing said anucleated blood cell-extracted nucleic acid fraction for the presence of a foetal disease or condition marker, wherein said foetal disease or condition marker is a foetal disease or condition-specific nucleic acid sequence or mutation in the nucleic acid of said foetus, or wherein said foetal disease or condition marker is a foetal disease or condition-specific expression profile of genes of a cell of said foetus.   
     
     
         2 . The method of  claim 1 , wherein said anucleated blood cells are thrombocytes or erythrocytes, preferably thrombocytes. 
     
     
         3 . The method of  claim 1 , wherein said foetal disease or condition is selected from the group consisting of congenital and genetic disorders. 
     
     
         4 . The method of  claim 3  wherein said genetic disorder is gene mutation of trisomy or wherein said foetal disease or condition is the foetal gender. 
     
     
         5 . The method of  claim 1 , wherein said foetal disease or condition marker is a nucleic acid of a foetus. 
     
     
         6 . The method of  claim 1 , wherein said expression profile is based on ribonucleic acid (RNA), preferably mRNA in said anucleated blood cell. 
     
     
         7 . The method of  claim 1 , wherein said step b) of analysing said anucleated blood cell-extracted nucleic acid fraction for the presence of a foetal disease or condition marker comprises the selective amplification of
 i) at least a part of said nucleic acid by (reverse transcriptase) polymerase chain reaction amplification using at least one foetal disease or condition marker-specific amplification primer or probe, or   ii) a plurality of mRNAs (or at least a part of the mRNAs) by reverse transcriptase polymerase chain reaction amplification to determine the expression level of the chromosomal genes encoding said mRNAs to thereby provide an expression profile for said genes and comparing said expression profile to a reference profile.   
     
     
         8 . The method of  claim 1 , wherein said method is part of a method of diagnosing said foetal disease or condition in a subject, and wherein the presence of said foetal disease or condition marker in said anucleated blood cell-extracted nucleic acid fraction is indicative of said foetus suffering from or at risk of suffering from said foetal disease or condition. 
     
     
         9 . A method for determining the stage of foetal disease or condition or the efficacy of a foetal disease or condition treatment in a subject, comprising the steps of:
 analysing a blood sample of a subject for the presence of a foetal disease or condition marker using the method according to  claim 1  at a first time point to thereby provide a first value for the level of said foetal disease or condition marker in said subject,   analysing a blood sample of said subject for the presence of a foetal disease or condition marker using the method according to any one of  claims 1 - 8  at a second time point to thereby provide a second value for the level of said foetal disease or condition marker in said subject, wherein said subject or said foetus has been subjected to a foetal disease or condition treatment between said first and second time point, and   comparing said first and second value to determine the efficacy of said foetal disease or condition treatment in said subject.   
     
     
         10 . A method for determining the stage of a foetal disease or condition in a subject, comprising the steps of:
 analysing a blood sample of a subject carrying a foetus for the presence of a foetal disease or condition marker using the method according to  claim 1  to thereby provide a test value for the level of said foetal disease or condition marker in said subject,   providing a reference value for the level of said foetal disease or condition marker wherein said reference value is correlated to a particular stage of foetal disease or condition, and   comparing said test and reference value to determine the stage of foetal disease or condition in said subject.   
     
     
         11 . A kit of parts adapted for performing the method recited in  claim 1 , the kit comprising a packaging material which comprises at least one of:
 a container for holding anucleated blood cells separated from a blood sample of a subject;   an agent for extracting nucleic acids from said anucleated blood cells;   an agent for selectively amplifying from said nucleic acids extracted from said anucleated blood cells a foetal disease or condition agent-specific nucleic acid sequence or a foetal disease or condition-specific gene expression profile of chromosomal genes of said subject, and   a printed or electronic instruction for performing the method recited in  claim 1 ,   
       the kit further comprising:
 a reference for said foetal disease or condition marker, wherein said reference is indicative for the presence or absence of said foetal disease or condition marker in said anucleated blood cells-extracted nucleic acid fraction. 
 
     
     
         12 . The kit of  claim 11 , wherein said reference is a reference value for the level of nucleic acids comprising said foetal disease or condition agent-specific nucleic acid sequence in anucleated blood cells in a healthy control subject or in a control subject suffering from said foetal disease or condition, or
 wherein said reference is a reference expression profile for said plurality of mRNAs in anucleated blood cells from a healthy control subject or from a control subject suffering from said foetal disease or condition.   
     
     
         13 . A kit of  claim 11 , wherein said agent is selected from a particle or fluorescent marker-labeled anti-anucleated blood cell antibody, or wherein said instruction is selected from an instruction for bead-based anucleated blood cells isolation, an instruction for FACS sorting of anucleated blood cells, an instruction for anucleated blood cell recovery by centrifugation, or negative selection of non-anucleated blood cell components. 
     
     
         14 . A device for diagnosing foetal disease or condition in a subject, the device comprising a support and at least one agent for specifically determining a level and/or activity of at least one nucleic acid in an anucleated blood cell sample of the subject attached to said support, and
 a computer-readable medium having computer-executable instructions for performing the method recited in  claim 1 .   
     
     
         15 . The device of  claim 14 , wherein said at least one agent is an oligonucleotide probe or sequencing primer. 
     
     
         16 . The device of  claim 14 , comprising a lateral flow device, a dipstick or a cartridge for performing a nucleic acid hybridization reaction between:
 an anucleated blood cells-extracted nucleic acid and at least one foetal disease or condition agent-specific amplification primer or oligonucleotide probe, wherein said foetal disease or condition agent-specific amplification primer or oligonucleotide probe hybridizes specifically to a foetal disease or condition agent-specific nucleic acid sequence, or   an anucleated blood cells-extracted nucleic acid and a plurality of gene-specific amplification primers or oligonucleotide probes for providing an disease-specific gene expression profile, wherein said genes are genes of a nucleated cell of the subject in which the foetal disease or condition is to be diagnosed.